Project 3: Sterol Transport Pathways in Cardiovascular Disease ABSTRACT The objective of Project 3 is to define fundamental mechanisms that regulate cellular lipid flux and to elucidate their impact on systemic metabolism. Dissecting signaling pathways that govern how cells store, transport, and metabolize lipids is expected to uncover new opportunities for therapeutic intervention in metabolic disease. Although nonvesicular cholesterol transport has long been hypothesized to be critical for lipid homeostasis in mammalian cells, the underlying mechanisms have remained obscure. We have discovered a novel transporter called Aster-B that appears to fill this important gap in our understanding of sterol transport. Aster-B is a previously uncharacterized protein that facilitates the direct transport of cholesterol from the plasma membrane (PM) to the ER. We propose a series of molecular, cell biological, and mouse studies to investigate the roles of the Aster-B cholesterol transport pathway in physiology and disease. Aim 1 is to elucidate the role of Aster-B in cellular cholesterol transport, efflux, and esterification. We identified Asterb as a novel cholesterol-responsive LXR target gene. Gain or loss of Aster-B alters cholesterol distribution and impairs cholesterol ester synthesis in response to cholesterol loading. Using biochemical approaches and complementary imaging modalities including electron and live-cell microscopy, we will define the mechanism of action of Aster-B and its role in macrophage sterol flux. Aim 2 is to determine the impact of Aster-B on sterol transport in vivo. Preliminary data indicate that Asterb is most highly expressed in macrophages, adrenal gland, and gonads. We will determine the effect of loss of Aster-B expression on whole-body and tissue-specific lipid homeostasis. We hypothesize that Aster-B is a critical mediator of cellular cholesterol transport downstream of the HDL receptor SR-BI. Aim 3 is to define the contribution of the macrophage Aster pathway to atherosclerosis. The LXR pathway is one of the strongest known determinants of atherosclerotic lesion development. Our observation that Aster-B expression is regulated by LXRs suggests that Aster-dependent cholesterol transport may impact macrophage foam cell formation and the development of atherosclerosis. We will test the impact of gain or loss of Aster function on macrophage cholesterol uptake and efflux. We will perform bone marrow transplant studies into LDLR-deficient mice to test the impact of Aster-B deficiency on lesion formation. Aim 4 is to identify additional components of the Aster pathway. We will perform protein-interaction screens using a biotin proximity labeling strategy. We will perform a chemoproteomic screen using HDL particles loaded with a cholesterol-mimetic probe that can be crosslinked to proteins and retrieved using a click-chemistry handle. This application leverages the unique and complementary strengths of each member of our PPG, bringing together a range of approaches and technological capabilities that would be unavailable to any single investigator. Understanding the molecular pathways that control cholesterol movement in macrophages is central to the overall theme of this PPG application and will advance our understanding of both physiology and pathophysiology.